Antimicrobial peptide isolated from halocynthia aurantium

ABSTRACT

The present invention relates to an antimicrobial peptide isolated from  Halocynthia aurantium , more particularly, to an antimicrobial peptide isolated from the body fluid of  Halocynthia aurantium  and an antimicrobial agent comprising the same as an active ingredient. The antimicrobial peptide of the present invention shows excellent antimicrobial activity under strong acidic and basic environments. Moreover, it also shows strong antimicrobial activity against resistant bacteria. So, it can be used usefully as a natural antimicrobial agent.

This application is the U.S. National Phase of International ApplicationPCT/KR2002/002195, international filing date of Nov. 22, 2002.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

This application includes a “SequenceListing.txt”, 7,680 bytes, updatedon Aug. 18, 2008, and submitted electronically via EFS-Web which ishereby incorporated by reference in its entirety. The submission of thesequence listing text file does not include any new matter.

FIELD OF THE INVENTION

The present invention relates to an antimicrobial peptide isolated fromHalocynthia aurantium, more particularly, to an antimicrobial peptideisolated from the body fluid of Halocynthia aurantium and anantimicrobial agent comprising the same as an active ingredient. Theantimicrobial peptide of the present invention shows excellentantimicrobial activity under strong acidic and basic environments.Moreover, it also shows strong antimicrobial activity against resistantbacteria. So, it can be used usefully as a natural antimicrobial agent.

BACKGROUND

Most researchers studying immunology have been interested rather inadaptive immunity having memory and specificity than in innate immunity,so far. Nevertheless, innate immunity plays an important role inself-defense system of animals. For instance, 1) innate immune cellsprohibit the invasion of microorganisms through skin or epitherial cellsof the intestines, 2) innate immune cells restrain pathogens invadinginto blood or body fluid with their phagocytosis, 3) innate immunitypreferentially prevents various invading microorganisms from growing inbody fluid after infection even before adaptive immunity or phagocytosisis activated since innate immunity does not have specificity. Cells thatare responsible for innate immunity use various antimicrobial substancessuch as simple inorganic compounds (H₂O₂, NO, etc), antimicrobialpeptides and proteins in order to function as the above. Antimicrobialpeptides or proteins have been reported to be on mucosal epithelialsurface, in body fluid and in intracellular organelles of phagocytes,and to have various sizes, structures and activity (Hancock, R. E. etal., Proc. Natl. Acad. Sci., 2000, 97, 8856-8861). But there are commoncharacteristics, too, that is, most antimicrobial peptides or proteinshave complementary positive charge to negative charge of cell membraneof microorganisms, antimicrobial proteins having enzyme activity(proteases or muramidases) hydrolyze the membrane of bacteria andantimicrobial peptides also target in cell membrane of microorganisms(Zhang, L. et al., J. Biol. Chem., 2001, 276, 35714-35722). Owing tothese mechanisms, antimicrobial peptides are expected to be very helpfulfor the development of novel antibiotics that can be effectively usedfor the bacteria having resistance against conventional antibiotics.Frequent appearance of resistant strains resulted from overuse ofchemical synthetic antibiotics evokes the interest in theseantimicrobial peptides as well.

Antimicrobial peptides are largely classified two groups: one group iscomposed of peptides having bipolar α-helical structure and the othergroup is composed of peptides having β-sheet structure stabilized byintradisulfide bonds. Cysteine containing antimicrobial peptides mostlykeep even number of cysteine residues from 2 to 8, which contribute tobuild intradisulfide bonds, resulting in the completion of a stablestructure. Table 1 shows a classification of antimicrobial peptideshaving β-sheet structure by the number of cysteine residue in amolecule.

TABLE 1 β-sheet antimicrobial peptides classified by the number ofintramolecular systeine Cysteine number Peptide Origin Reference 2Dodecapeptide Ruminants 1 Thanatin Insects 2 Bombinin Amphibian 3 4Tachyplesin Tachypleus 4 tridentatus Androctonin Scorpion 5 ProtegrinPig 6 6 α-defensin Leucocytes of 7 mammalian β-defensin Epithelial 8cells of mammalian 8 Hepcidin Human liver 9 1. Romeo, D. et al., J.Biol. Chem., 1988, 263, 9573-9575. 2. Fehlbaum, P. et al., Proc, Natl,Acad, Sci., 1996, 93, 1221-1225. 3. Goraya, J. et al., Eur. J. Biochem.,2000, 267, 894-900. 4. Iwanaga, S. et al., J. Biochem., 1998, 123, 1-15.5. Hetru, C. et al., Biochem. J., 2000, 345, 653-644. 6. Ganz, T. etal., Drugs, 2000, 9, 1731-1742. 7. Lehrer, R. I. et al., Annu. Rev.Immunol., 1993, 11, 105-128. 8. O'Neil, D. A. et al., J. Immunol., 1999,163, 6718-6724. 9. Krause, A. et al., FEBS Lett., 2000, 480, 147-150.

Working mechanism and specificity of antimicrobial peptides depend onthe way to work mutually with bacterial cell membranes. Generally,peptides are accepted through self-promoted uptake pathway by workingwith LPS (lipopolysaccharide) on the surface of Gram-negative bacteria.The first step of the accepting process is that the peptides are adheredto divalent cation-binding sites of LPS on cell surface, and the secondstep is that the peptides are inserted in cell membrane to form achannel.

In the first step, peptides can bind to LPS with 3 times as highaffinity as divalent cations like Mn⁺⁺ or Mg⁺⁺, so that they can besubstituted for the divalent cations, causing a break down of a generalproperty of cell membrane, especially of outer membrane. Such affectedbacterial cell membrane makes a gap temporarily, through whichhydrophobic substances, low-molecular proteins or antibiotics can passand especially peptides are inserted effectively (Piers, K. L. et al.,Antimicrob. Agents Chemother., 1994, 38, 2311-2316).

In the second step, peptides are inserted in cell membrane to form achannel, during which magnetism of cation peptides works with anions ofbacterial membrane, so that hydrophobic region faces membrane andhydrophilic region faces inner side to form a channel (Hancock, R. E. etal., Adv. Microbial Physiol., 1995, 37, 135-175). The channel is formedwell when potential difference is big, the amount of anion lipids isgreat and the quantity of cholesterol is small. A well-formed channelcauses a break down of membrane structure, resulting in the death ofbacteria (Falla, T. et al., J. Biol. Chem., 1996, 271, 19298-19303). Onthe contrary, eukaryotic cells containing a huge amount of cholesterolbut a small quantity of anion lipid do not provide a good condition forthe working of peptides. Thus, the peptides show a highly selectiveactivity against bacteria. Based on the above reasons, antimicrobialpeptides are noticed as novel antibiotics with less cytotoxicity.Besides, the advantages of antimicrobial peptides, as novel antibiotics,are as follows.

1. Preventing the appearance of resistant bacteria by destroyingbacterial membrane physically.

2. Working faster than the life cycle of bacteria.

3. Working effectively on resistant bacteria having resistance againstconventional antibiotics.

4. Having wide antimicrobial spectrum.

5. Having an anti-endotoxicity effect owing to the binding capacity toLPS, etc.

6. Being able to be mass-produced using genetic engineering techniquesand developed as a novel medicine with a less production cost.

From the viewpoint of animal systematic taxonomy, a tunicate belongingto deuterostomia is a kind of invertebrates classified intoprotochordata with such characteristics as having notochord and dorsaltubular nerve cord during tadpole larva period. Thus, a tunicate can beclassified in pre-vertebrata with respect to systematic evolutionistics.Owing to such taxonomical position, a tunicate has been regarded as amodel animal to prove evolutional origin of animal immune system.Especially, the body cavity (hemocoel) of a tunicate was observed tohave lots of phagocytes having similar forms and functions togranulocytes and macrophages found in circulatory system of vertebrata(Bone, Q., The Origin of Chordates, 1979, 2nd edn). Studies to detectout antimicrobial peptides from body fluid cells of a tunicate have beenundergoing and clavanin (Lee, I. H. et al., FEBS Lett., 1997, 400,158-162; Lee, I. H. et al., Infection and Immunity, 1997, 65, 2898-2903;Zhao, C. et al., FEBS Lett., 1997, 410, 490-492) and styelin (Lee, I. H.et al., Comp. Biochem. Physiol. B Biochem. Mol. Biol., 1997, 118,515-521; Zhao, C. et al., FEBS Lett., 1997, 412, 144-148) separated frombody fluid cells of Styela clava are the representative antimicrobialpeptides found out so far.

Two kinds of tunicates inhabit in the country. One is Halocynthiaroretzi inhabiting mainly in southwest seashores or raised artificiallyand the other is Halocynthia aurantium, also called “silky sea squirt”,inhabiting only in Sokcho (Kangwon-Do, Korea) area of the east coast.The former has been studied in Japan many years and disclosed to have anantimicrobial substance in the shape of transformed peptide(tetrapeptide) like halocyamine (Azumi, K. et al., Experientia, 1990,46, 1066-1068; K. Azumi et al., Biochemistry, 1990, 29, 159-165). Butthere is no report yet that an antimicrobial peptide is separated fromthe latter, Halocynthia aurantium.

Thus, the present inventors investigated if there is any antimicrobialpeptide in body fluid cells of Halocynthia aurantium. As a result, thepresent inventors separated an antimicrobial peptide named asdicinthaurin (Lee, I. H. et al., Biochem. Biophys. Acta, 2001, inpress), and further, separated another antimicrobial peptide recently.The present inventors have accomplished this invention by analyzing thestructure of the newly separated antimicrobial peptide and confirmingthe excellent antimicrobial activity thereof.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

It is an object of the present invention to provide an antimicrobialpeptide isolated from Halocynthia aurantium and an antimicrobial agentcomprising the same as an active ingredient.

To achieve the above object, the present invention provides anantimicrobial peptide isolated from the body fluid of Halocynthiaaurantium and an antimicrobial agent comprising the same as an activeingredient.

Hereinafter, the present invention is described in detail.

The present invention provides an antimicrobial peptide isolated fromthe body fluid of Halocynthia aurantium.

The antimicrobial peptide of the present invention is isolated fromtunicate, wherein the tunicate is preferred to be Halocynthia aurantiumcalled “silky sea squirt”. However, it is a common knowledge for thepeople in this field that the antimicrobial peptide of the presentinvention is not limited thereto and can be isolated from otherorganisms or synthesized artificially.

The present invention provides a peptide represented by <ChemicalFormula 1>having 18 amino acids represented by each figures.W₁X₂B′₃U₄X₅X₆B₇B₈U₉X₁₀B′₁₁C₁₂U₁₃B₁₄U₁₅X₁₆x₁₇u₁₈ (SEQ lDNO:11).  <Chemical Formula 1>

In the above <Chemical Formula 1>,

W represents tryptophane or its derivatives;

X (X₂, X₅, X₆, X₁₀, X₁₆ or X₁₇) represents more than one amino acidresidue selected from a group consisting of tyrosine, valine,isoleucine, leucine, methionine, phenylalanine and tryptophane, and thederivatives thereof;

B (X₇, X₈ or X₁₄) represents more than one amino acid residue selectedfrom a group consisting of arginine, lysine and histidine, and thederivatives thereof;

B′ (X₃ or X₁₁) represents more than one amino acid residue selected froma group consisting of arginine, lysine and histidine or from a groupconsisting of asparagine and glutamine, and the derivatives thereof; and

U (X₄, X₉, X₁₃, X₁₅ or X₁₈) represents more than one amino acid residueselected from a group consisting of glycine, serine, alanine andthreonine, and the derivatives thereof.

As for the peptide of the present invention represented by the above<Chemical Formula 1>, it is preferable to select tryptophane for W,select one from a group consisting of leucine, isoleucine and valine forX, one from a group consisting of asparagine, glutamine, histidine,lysine and arginine for B, one from a group consisting of alanine,serine, and glycine for U, and select cysteine for C (SEQ ID NO: 12).

For building the peptide of the present invention represented by theabove <Chemical Formula 1>, it is more preferable to select tryptophanefor W₁, leucine for X₂, asparagine for B′₃, alanine for U₄, leucine forX₅, leucine for X₆, histidine for B₇, histidine for B₈, glycine for U₉,leucine for X₁₀, asparagine for B′₁₁, cysteine for C₁₂, alanine for U₁₃,lysine for B₁₄, glycine for U₁₅, valine for X₁₆, leucine for X₁₇ andalanine for U₁₈. Thus, it is most preferable for the peptide of thepresent invention to have amino acid sequence represented by SEQ. ID. No1.

The present invention also provides a peptide having 15 amino acidsrepresented by <Chemical Formula 2> in which three amino acids (W₁X₂B′₃)at N-terminal of the peptide represented by the above <Chemical Formula1> are lost.U₄X₅X₆B₇B₈U₉X₁₀B′₁₁C₁₂U₁₃B₁₄U₁₅X₁₆X₁₇U₁₈ (SEQ ID NO:13).  <ChemicalFormula 2>

In the above Formula,

X (X₂, X₃, X₇, X₁₃ or X₁₄) represents more than one amino acid residueselected from a group consisting of tyrosine, valine, isoleucine,leucine, methionine, phenylalanine and tryptophane, and the derivativesthereof;

B (X₄, X₅ or X₁₁) represents more than one amino acid residue selectedfrom a group consisting of arginine, lysine and histidine, and thederivatives thereof;

B′ (X₈)represents more than one amino acid residue selected from a groupconsisting of arginine, lysine and histidine or from a group consistingof asparagine and glutamine, and the derivatives thereof; and

U (X₁, X₆, X₁₀, X₁₂ or X₁₅) represents more than one amino acid residueselected from a group consisting of glycine, serine, alanine andthreonine, and the derivatives thereof.

As for the peptide of the present invention represented by the above<Chemical Formula 2>, it is preferable to select one from a groupconsisting of leucine, isoleucine and valine for X, one from a groupconsisting of asparagine, glutamine, histidine, lysine and arginine forB, one from a group consisting of alanine, serine, and glycine for U,and select cysteine for C (SEQ ID NO: 14).

For building the peptide of the present invention represented by theabove <Chemical Formula 2>, it is more preferable to select alanine forU₄, leucine for X₅, leucine for X₆, histidine for B₇, histidine for B₈,glycine for U₉, leucine for X₁₀, asparagine for B′₁₁, cysteine for C₁₂,alanine for U₁₃, lysine for B₁₄, glycine for U₁₅, valine for X₁₆,leucine for X₁₇ and alanine for U₁₈ (SEQ ID NO:15). Thus, it is mostpreferable for the peptide of the present invention to have amino acidsequence represented by SEQ. ID No 2.

The present invention further provides a peptide in dimer formrepresented by <Chemical Formula 3> wherein the cysteine residues of twopeptides, each represented by <Chemical Formula 1> (SEQ ID NO:11), arecombined with each other by disulfide bond.

The above amino acids represented by each figures are the same asrepresented by <Chemical Formula 1> and the peptide is most preferablyformed by combining two amino acids of peptides represented by SEQ. ID.No 1, the 12^(th) amino acid each, together by disulfide bond.

The present invention also provides a peptide in dimer form representedby <Chemical Formula 4> wherein the cysteine residues of two peptides,each represented by <Chemical Formula 2> (SEQ ID NO:13), are combinedwith each other by disulfide bond.

The above amino acids represented by each figures are the same asrepresented by <Chemical Formula 2> and the peptide is most preferablyformed by combining two amino acids of peptides represented by SEQ. ID.No 2, the 9^(th) amino acid each, together by disulfide bond.

The present invention also provides a peptide in dimer form representedby <Chemical Formula 5> wherein the cysteine residue of the peptiderepresented by <Chemical Formula 1> (SEQ ID NO:11) is combined with thatof the peptide represented by <Chemical Formula 2> (SEQ ID NO:13) bydisulfide bond.

The above amino acids represented by each figures are the same asrepresented in <Chemical Formula 1> and <Chemical Formula 2>. For thepeptide above, it is most preferable to combine the 12^(th) amino acidof the peptide represented by SEQ. ID. No 1 and the 9^(th) amino acid ofthe peptide represented by SEQ. ID. No 2 by disulfide bond.

In the preferred embodiment of the present invention, the presentinventors separated a peptide represented as <Chemical Formula 5> frombody fluid of Halocynthia aurantium and confirmed that the peptide hadantimicrobial activity. We, the present inventors named the peptide“halocidin”. By detecting out the structure of halocidin, the presentinventors confirmed that halocidin consisted of peptides represented as<Chemical Formula 1> and <Chemical Formula 2> in which cysteine residueswere combined each other by disulfide bond (see FIG. 5). Named a peptiderepresented as <Chemical Formula 1>“18Hc” and a peptide represented as<Chemical Formula 2>“15Hc”. In order to analyze the characteristics ofhalocidin, the present inventors prepared peptides in dimer form eachrepresented as <Chemical Formula 3> and <Chemical Formula 4> usingpeptides represented as <Chemical Formula 1> and <Chemical Formula 2>,and then named them “di-18Hc” and “di-15Hc”.

The inventors also named a peptide wherein a C-terminal amino acid waseliminated from 18Hc “(18-1)Hc” and a peptide wherein two C-terminalamino acids were eliminated “(18-2)Hc”. In the same manner, Peptideswherein 3 to 6 C-terminal amino acids were removed were named“(18-3)Hc”, “(18-4)Hc”, “(18-5)Hc” and “(18-6)Hc” respectively. Apeptide wherein all histidine residues were substituted with lysine wasnamed “Hck”. When lysine was added to N-terminal of a peptide, theletter “K” was added in the first place of the name of the peptide andthe added number of lysine was marked as (+) next to the number. Forexample, as one lysine was added to N-terminal of 18Hc, it was named“K(18+1)Hc”.

The mass of peptides represented as <Chemical Formula 1 to 5> wasmeasured, resulting in 1,929 Da, 1,516 Da, 3,861 Da, 3,031 Da and 3,445Da, respectively (see Table 3). Especially, pI value of halocidin, apeptide represented as <Chemical Formula 5>, was 8.965 and halocidin wasa peptide in hetero-dimer form having helical wheel structure (see FIG.8).

In order to confirm if the peptides of the present invention representedas <Chemical Formula 1 to 5> have antimicrobial activity, performedradical diffusion analysis (see FIG. 9), colony counting analysis (seeFIG. 10), hemolysis analysis (see FIG. 11), radical diffusion analysison Gram negative strain (see FIG. 12, FIG. 13 and FIG. 14) and radicaldiffusion analysis on Gram positive strain (see FIG. 15, FIG. 16 andFIG. 17). As a result, it was disclosed that those peptides representedas <Chemical Formula 1 to 5> had great antimicrobial activity.Precisely, peptides in dimer forms represented as <Chemical Formula 2, 4and 5> had greater antimicrobial activity and a peptide represented as<Chemical Formula 2> showed the greatest antimicrobial activity, aboveall.

In order to confirm if the peptides of the present invention still keepantimicrobial activity under strict conditions in vivo, measuredantimicrobial activity of the peptides under the condition of pH 5.5that was the same acidic condition as the environment in epithelialcells, urethra, vagina, etc, and NaCl 200 mM that was higher than basiccondition of intra-blood (NaCl 150 mM). As a result, it was confirmedthat a peptide represented as <Chemical Formula 5> showed antimicrobialactivity under the condition of pH 5.5-pH 7.4 (see Table 4) and NaCl 100mM-NaCl 200 mM as well (see Table 5).

Based on the above results, the peptides of the present inventionrepresented as <Chemical Formula 1 to 5> were proved to have excellentantimicrobial activity, comparing to the conventional antibiotics.Particularly, the peptides were confirmed to have excellentantimicrobial activity under strong acidic and basic environments, andhave strong antimicrobial activity against resistant bacteria.

The present invention further provides an antimicrobial agent containingthe above-mentioned peptide as an active ingredient.

As explained hereinbefore, the peptide of the present invention has anexcellent antimicrobial activity under strong acidic and basicenvironments. So, it can be effectively used as an antimicrobial agent.

Thus, the peptide of the present invention can be included as an activeingredient for preparing an antimicrobial agent. The antimicrobial agentof the present invention can be administered orally or parenterally andbe used in general form of pharmaceutical formulation.

The antimicrobial agent of the present invention can be prepared fororal or parenterally administration by mixing with generally-usedfillers, extenders, binders, wetting agents, disintegrating agents,diluents such as surfactant, or excipients. Solid formulations for oraladministration are tablets, pill, dusting powders and capsules. Thesesolid formulations are prepared by mixing one or more suitableexcipients such as starch, calcium carbonate, sucrose or lactose,gelatin, etc with one or more halocidin. Except for the simpleexcipients, lubricants, for example magnesium stearate, talc, etc, canbe used. Liquid formulations for oral administrations are suspensions,solutions, emulsions and syrups, and the abovementioned formulations cancontain various excipients such as wetting agents, sweeteners, aromaticsand preservatives in addition to generally used simple diluents such aswater and liquid paraffin. Formulations for parenteral administrationare stirilized aqueous solutions, water-insoluble excipients,suspensions, emulsions, and suppositories. Water insoluble excipientsand suspensions can contain, in addition to the active compound orcompounds, propylene glycol, polyethylene glycol, vegetable oil likeolive oil, injectable ester like ethylolate, etc. Suppositories cancontain, in addition to the active compound or compounds, witepsol,macrogol, tween 61, cacao butter, laurin butter, glycerinated gelatin,etc.

In general, it has proved advantageous both in human and in veterinarymedicine to administer the active compound or compounds according to thepresent invention in total amounts of about 0.5 mg/kg to about 1 mg/kg,preferably 0.1-0.5 mg/kg of body weight, one to two times every 24hours, if appropriate, in the form of several individual doses, toachieve the desired results.

The antimicrobial agent of the present invention can be used widely asan antibacterial agent or an antiviral agent to control virus, Grampositive bacteria, Gram negative bacteria, fungi, yeast and protozoaharming plants, animals and human. The antimicrobial agent of thepresent invention can be used either independently or together withother antibiotics such as erythromycin, tetracycline, azithromycin,vancomycin, cephalosporins, etc. Further, the antimicrobial agent of thepresent invention can also be used as food additives, cosmetics,ointments, injections, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is an AU-PAGE photograph showing the purification process ofhalocidin from Halocynthia aurantium;

Lane 1: Acidic extracts of body fluid cells of Halocynthia aurantium,

Lane 2: 51^(st)-81^(st) fractions passed through Sepadex G-50 column,

Lane 3: 35^(th)-45^(th) fractions passed through Prep. AU-PAGE, and

Lane 4: Halocidin purified by RP-HPLC

FIG. 2 is a graph showing the result of C18 RP-HPLC with purifiedhalocidin;

FIG. 3 is a photograph showing the result of SDS-PAGE with purifiedhalocidin;

Lane M: Standard molecular weight marker,

Lane 1: Natural halocidin, and

Lane 2: Halocidin cut by dithiothreitol

FIG. 4 is a graph showing the result of MALDI mass analysis withpurified halocidin;

FIG. 5 is a diagram showing the structure and amino acid sequence of thetwo constituents of halocidin;

FIG. 6 is a set of graphs showing the result of RP-HPLC with a naturalhalocidin and a synthetic halocidin;

A: 15Hc and 18Hc,

B: di-15Hc, di-18Hc and halocidin,

C: Natural halocidin

FIG. 7 is a set of graphs showing the CD spectra of 18Hc suspended inphosphate buffer (pH 7.4) (pink line), 20 mM SDS phosphate buffer (pH7.4) (black line) and 10 mM phosphate buffer containing 50% (v/v)trifluoroethanol (pH 7.4)(red line);

A: 18Hc, B: di-18Hc

FIG. 8 is a set of a photograph and a graph showing the helical diagram(A) and pI (B) of halocidin (18Hc);

FIG. 9 is a set of photographs and graphs showing the result of radicaldiffusion analysis of a peptide affecting MRSA (A and C) and MDRPA (Band D);

a: 15Hc,

b: di-15Hc,

c: 18Hc,

d: di-18Hc,

e: Halocidin,

f: Magainin 1,

g: Bufforin 2

FIG. 10 is a set of graphs showing the antimicrobial activity of apeptide measured by colony counting analysis;

FIG. 11 is a graph showing the result of hemolytic assay with a peptide;

FIG. 12 is a set of graphs comparing antimicrobial activities of 15Hc,18Hc, halocidin, bufforin 2 and magainin 1 to Gram-negative bacteria;

A: Pseudomonas aeruginosa,

B: Salmonella cholerasuis,

C: Salmonella parotyphi A,

D: E. coli K112 and

E: E. coli DH5α

FIG. 13 is a set of graphs comparing antimicrobial activities of(18-1)Hc, di-(18-2)Hc, 18Hc and di-18Hc to Gram-negative bacteria;

A: Pseudomonas aeruginosa,

B: Salmonella cholerasuis,

C: Salmonella parotyphi A,

D: E. coli K112 and

E: E. coli DH5α

FIG. 14 is a set of graphs comparing antimicrobial activities of(18-2)Hc, di-(18-2)Hc,

(18)Hck and di-(18)Hck to Gram-negative bacteria;

A: Pseudomonas aeruginosa,

B: Salmonella cholerasuis,

C: Salmonella parotyphi A,

D: E. coli K112 and

E: E. coli DH5α

FIG. 15 is a set of graphs comparing antimicrobial activities of 15Hc,18Hc, halocidin, bufforin 2 and magainin 1 to Gram-positive bacteria;

A: Staphylococcus aureus,

B: Micrococcus luteus,

C: Enterococcus faecalis,

D: Bacillus subtilus and

E: MRSA

FIG. 16 is a set of graphs comparing antimicrobial activities of(18-1)Hc, di-(18-1)Hc, 18Hc and di-18Hc to Gram-positive bacteria;

A: Staphylococcus aureus,

B: Micrococcus luteus,

C: Enterococcus faecalis,

D: Bacillus subtilus and

E: MRSA

FIG. 17 is a set of graphs comparing antimicrobial activities of(18-2)Hc, di-(18-2)Hc,

(18)Hck and di-(18)Hck to Gram-positive bacteria;

A: Staphylococcus aureus,

B: Micrococcus luteus,

C: Enterococcus faecalis,

D: Bacillus subtilus and

E: MRSA

EXAMPLES

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Isolation of an Antimicrobial Peptide from Halocynthiaaurantium

<1-1> Isolation of Acid Extract from Halocynthia aurantium

Bought Halocynthia aurantium, called silky sea squirt, at a fish marketin Sockcho, Kangwon-Do, Korea, washed the outer skin alive with 70%ethanol, and dried thereof. Cut the exhalent opening of the driedtunicates crossways and put thereof into a 50 ml tube containing 150 mgof EDTA to collect hemolymph. Removed impurities from the obtainedhemolymph using 74 μm pore sized mesh filter (Netwell, Corning Costar,Cambridge, Mass., USA), after which centrifuged thereof at 4° C. with300 g for 30 minutes. After centrifugation, suspended precipitated bodyfluid cells, hemocytes, in 30 ml of 0.34 M sucrose solution andcentrifuged again at 4° C. with 300 g for 30 minutes. On completingcentrifugation, isolated newly formed cell layer and suspended thereofin 10 ml of cooled 5% acetic acid solution. After sonication with theabove suspension 5 times 15 seconds each, added 40 ml of 5% acetic acidsolution thereto. Mixed the solution extracted by acetic acid at 4° C.for overnight, followed by centrifuging at 4° C. with 20,000 g for 30minutes. Used the obtained supernatants from centrifugation as a testmaterial for purifying antimicrobial peptides.

Quantified the protein of acid extracts from Halocynthia aurantium usingbicinchoninic acid (Sigma) and obtained eluting fractions by loading thesupernatants containing at least 50 mg of protein to Sephadex G-50 gelfiltering column (Sigma) equilibrated with 5% acetic acid solution.

<1-2> Antimicrobial Activity of Acid Extracts of Halocynthia aurantium

In order to measure the antimicrobial activity of acid extracts ofHalocynthia aurantium obtained in the above Example <1-1>, the presentinventors performed ultrasensitive radial diffusion assay. Particularly,analyzed 150 fractions eluted in the above Example <1-1> 5 times each.Took 100 μl from 2 ml of each fraction and concentrated thereof with avacuum centrifugation (Centra Evaporator, Bioneer, Korea), which wassuspended in 5 μl of 0.01% acetic acid solution. Meanwhile, preparedagarose plate including wells 3 mm in diameter by adding methicillinresistance Staphylococcus aureus (referred as “MRSA” hereinafter, SeoulWomen's University, Korea CCARM3001) of mid-logarithmic phase to gelcomprising sterilized citrate phosphate buffer (9 mM sodium phosphate, 1mM sodium citrate, pH 7.4), 1% (w/v) type 1 agarose (lowelectroendosmosis agarose) (A 6013, Sigma) and 3% tryptic soy broth(TSB, Difco, Detroit, Mich., USA). Loaded the above fractions onto thewells of agarose plate containing the above bacteria. Reacted thereoffor 3 hours to make the peptides spread into the agarose gel. Added 10ml of overnutrition medium comprising 6% TSB and 1% agarose gel thereto.Cultured the above plate for overnight until the colonies of the abovebacteria were formed. Confirmed the antimicrobial activity of the loadedpeptide by measuring the diameter of clearing zone formed around theloaded peptide fraction.

As a result, the clearing zones formed around #51-#81 peptide fractionswere the biggest, suggesting that the fractions had high antimicrobialactivity.

<1-3> Purification of Peptide in Fractions Having Antimicrobial Activity

In order to purify peptide fractions having antimicrobial activity moreclearly, centrifuged #51-#81 fractions that were confirmed to haveantimicrobial activity in the pre-stage, concentrated and loaded thereofon preparative acid urea polyacrylamide gel electrophoresis (referred“Prep AU-PAGE” hereinafter). Adjusted the current speed of Prep AU-PAGEto 60 ml/hour and divided by 2 ml for a fraction.

Put Prep AU-PAGE fractions in 2 ml tubes and concentrated with a rotaryconcentrator. Performed electrophoresis with each fraction at intervalsof 10 numbers in two AU-PAGE gels. Stained one gel with Coomasie blue toconfirm bands and investigated the antimicrobial activity of proteinbands of the other gel with gel overlay assay using MRSA. For the geloverlay assay, put the electrophoresed gel on 10 ml of underlay agarcontaining MRSA and let it to be reacted at 37° C. for 3 hours in orderfor the peptides to diffuse into agarose gel, after which poured 10 mlof over-nutrition medium (6% TSB and 1% agarose gel).

As a result, proteins contained in #35-#45 fractions were confirmed tohave antimicrobial activity (FIG. 1).

In order to purify #35-#45 fractions that were confirmed to haveantimicrobial activity finally, loaded them to C18 reverse phase highperformance liquid chromatography (referred as “RP-HPLC” hereinafter)column (Vydac 218TP54: The Separation Group, Hesperia, Calif.). For thefirst 10 minutes after loading those samples, washed the column byspilling 5% acetonitrile containing 0.1% trifluoroacetic acid (TFA) atthe speed of 0.5 ml/minute. Thereafter, increased the concentration ofacetonitrile by 1%/minute for 60 minutes. During the process, collectedpeak fraction of each concentration of acetonitrile. Concentrated 10% ofeach collected fraction with a vacuum rotary concentrator, followed byconfirming antimicrobial activity with radial diffusion analysis.

As a result, confirmed that a peptide isolated at 50.2 minute at whichthe concentration of acetonitrile reached 45.2% had antimicrobialactivity and named it “halocidin” (FIG. 2).

Example 2 Analysis of Characteristics of Purified Halocidin

<2-1 Mass Analysis of Halocidin

In order to clarify characteristics of halocidin, a novel peptide havingantimicrobial activity, isolated from Halocynthia aurantium in the aboveExample 1, the present inventors performed SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) and AU-PAGE. Particularly,after freeze-drying halocidin, a peptide having antimicrobial activity,isolated in the above Example 1, added 25 μl of 8 M urea and 25 μl of0.4 M ammonium bicarbonate (pH 8.0) and melted. Added 45 mMdithiothreitol thereto and induced reaction at 50° C. for 15 minutes. Inorder to produce a vinylpyridine derivative of halocidin monomer, added15 μl of vinylpyridine to the above halocidin mixture and cooled thereofat room temperature, after which let the mixture to be reacted in thedark condition at least for 30 minutes. Extracted the final reactantswith RP-HPLC. Performed Tricine SDS-PAGE and AU-PAGE to confirm whetherthe product was correctly extracted, measured the molecular weight ofthe extracted halocidin with MALDI (matrix-associated laser desorptionionization) mass analyzer (Voyager-DE STR, PerSeptive Biosystems, USA)and analyzed amino acid sequences with a Gas-phase Edman degradationmethod using Procise 419 (Applied Biosystems, USA).

As a result, it was confirmed that the mass of halocidin extracted byRP-HPLC and halocidin monomers cut by dithiotreitol were 3.4 kDa and 1.5and 1.8 kDa respectively (FIG. 3), and major peak of halocidin was seenat the point of 3443.6836 m/z and minor peaks were seen at the points of1515.7487 m/z and 1929.9151 m/z (FIG. 4). The structure of halocidin wasalso confirmed in which cysteine residues of a monomer consisting of 18amino acids was combined with cysteine residues of another monomerconsisting of 15 amino acids by disulfide bond (FIG. 5).

Conclusively, the present inventors found out that halocidin, a peptidehaving antimicrobial activity isolated from Halocynthia aurantium, has3,443.7 Da molecular weight and is constructed by 15 monomer and 18monomer in which cysteine residues were combined each other by disulfidebonds. Finally, the present inventors named 18 monomer and 15 monomerforming halocidin “18Hc” and “15Hc” respectively.

<2-2> Preparation of Synthetic Halocidin and Comparing the Mass of theSynthetic Halocidin with that of Halocynthia aurantium Halocidin

After confirming the structure of halocidin formed by 15Hc and 18Hc, thepresent inventors prepared synthetic peptides in order to analyze andcompare the characteristics thereof. Particularly, artificiallysynthesized halocidin monomers using an automatic solid-phase peptidesynthesizer (Pioneer, Applied Biosystems, USA) and purified withRP-HPLC. Mixed 1 mg/ml of each synthesized peptide for homodimer orheterodimer (halocidin) formed by 15 monomer and 18 monomer in 0.1 Mammonium bicarbonate solution and let the mixture in the air over 72hours, resulted in the completion of synthetic peptides. Measured themass of synthesized monomers, homodimers and heterodimers using a MALDImass analyzer, by which confirmed if halocidin was correctlysynthesized.

The sequences of synthesized peptide monomers are shown in Table 2.

TABLE 2 Peptide Sequence Sequence list 18Hc WLNALLHHGLNCAKGVLA SEQ. ID.No 1 (18-1)Hc WLNALLHHGLNCAKGVL SEQ. ID. No 3 (18-2)Hc WLNALLHHGLNCAKGVSEQ. ID. No 4 (18-3)Hc WLNALLHHGLNCAKG SEQ. ID. No 2 (18-4)HcWLNALLHHGLNCAK SEQ. ID. No 5 (18-5)Hc WLNALLHHGLNCA SEQ. ID. No 6(18-6)Hc WLNALLHNGLNC SEQ. ID. No 7 (18)Hck WLNALLKKGLNCAKGVLA SEQ. ID.No 8 K(18 + 1)Hc KWLNALLHHGLNCAKGVLA SEQ. ID. No 9 K(18 + 1)HckKWLNALLKKGLNCAKGVLA SEQ. ID. No 10

As a result, it was confirmed that the expected masses of Halocynthiaaurantium halocidin and synthetic halocidin and the masses aftermeasuring with a MALDI mass analyzer were all the same (Table 3).

TABLE 3 MALDI Expected measured Peptide mass mass Halocynthia 15Hc1515.9 1515.74 aurantium 18Hc 1929.4 1929.91 halocidin Halocidin 3443.33443.68 Synthetic 15Hc 1515.9 1515.70 halocidin 18Hc 1929.4 1928.92di-15Hc 3029.8 3031.07 di-18Hc 3856.8 3861.06 Halocidin 3443.3 3445.04<2-3> Comparison of Elution Peaks of Halocynthia aurantium Halocidin andSynthetic Halocidin

The present inventors performed RP-HPLC to reconfirm eluting peaks ofHalocynthia aurantium halocidin and synthetic halocidin that had samemasses as seen in the above Example <2-2>. Particularly, loaded thesolution containing Halocynthia aurantium halocidin extracted byacetonitrile to RP-HPLC column and spilled 5% acetonitrile into thecolumn for 10 minutes (1 minute/ml). Measured the fractions eluted bythe time. As a result, two monomers (15Hc and 18Hc) of Halocynthiaaurantium were eluted at the 42^(nd) minute with 36.8% acetonitrileconcentration and at the 52^(nd) minute with 46.3% acetonitrileconcentration respectively (FIG. 6A). Meanwhile, two homodimers, di-15Hcand di-18Hc, were eluted from the fractions with 39.2% acetonitrileconcentration and with 51.7% acetonitrile concentration respectively.Heterodimers forming the structure of halocidin were eluted from thefractions with the same acetonitrile concentrations (FIG. 6C) as thecase of eluting natural halocidin (FIG. 6B).

<2-4> Identification of the Secondary Structure of Halocidin

In order to identify the secondary structure of halocidin, the presentinventors investigated CD spectra of the halocidin. Particularly,suspended 18Hc and di-18Hc in phosphate buffer (pH 7.4), 20 mM SDSphosphate buffer (pH 7.4) and 50% (v/v) trifluorethanol 10 mM phosphatebuffer (pH 7.4) at 25° C. using 1 mm rectangular cell. Measured circulardichroism spectrum using CJ-715 CD/ORD sepectropolaimeter (JASCO. Co).

As a result, 18Hc and di-18Hc were confirmed to have α-helix structurehaving maximum value at the point of 193 nm and two minimum values atthe points of 208 nm and 222 nm when being suspended in 20 mM SDSphosphate buffer and 50% (v/v) trifluorethanol 10 mM phosphate buffer(FIG. 7).

<2-5> Measurement of Helical Wheel Diagram and pI

In order to confirm the characteristics of halocidin more accurately,the present inventors measured helical wheel diagram and pI usingANTHEPROT 2000 V 5.2 software. As a result, confirmed the fact that 18Hchas a helical wheel structure and amphipathicity resulted fromclustering of polar and non-polar residues (FIG. 8A). Measured electriccharges of 18Hc by pH change, resulting in the confirmation that pI of18Hc is 8.965 (FIG. 8B).

Example 3 Analysis of Antimicrobial Activity of Halocidin

In order to analyze antimicrobial activity of a novel antimicrobialpeptide halocidin isolated from Halocynthia aurantium, the presentinventors performed ultra-sensitive radical diffusion assay, colonycounting assay, hemolytic assay and antimicrobial activity analysisagainst Gram-positive or Gram-negative bacteria.

<3-1> Ultra-Sensitive Radical Diffusion Assay

The present inventors performed ultra-sensitive radical diffusion assaywith synthetic peptides prepared in the above Example <2-2>.Particularly, measured antimicrobial activity of each 15Hc, di-15Hc,18Hc, di-18Hc, halocidin, magainin 1 (Sigma) (control group) and buforin2 (Sigma) (comparative group) against MRSA and multi drug resistancePseudomonas aeruginosa (referred as “MDRPA” hereinafter)(Seoul Women'sUniversity CCARM2002) according to the concentration of the peptides.

As a result, it was confirmed that buforin 2 that was known to have highantimicrobial activity to MRSA strain was proved to have antimicrobialactivity by that the diameter of clear zone was enlarged as theconcentration increased. 18Hc, di-18Hc and halocidin were confirmed tohave higher antimicrobial activity than buforin 2 as the concentrationincreased. Especially, di-18Hc showed the highest antimicrobial activity(FIG. 9A and FIG. 9C). Meanwhile, magainin 1 and buforin 2 were provedto have antimicrobial activity to MDRPA strain. And, 15Hc, di-15Hc,18Hc, di-18Hc and halocidin were proved to have higher antimicrobialactivity than comparative group (FIG. 9B and FIG. 9D).

Based on the above results, the present inventors confirmed thatHalocynthia aurantium halocidin has high antimicrobial activity, andespecially, peptides in the form of homodimer constructed by 15-monomerand 18-monomer, which are constituents of halocidin, have higherantimicrobial activity rather than halocidin itself.

<3-2> Colony Counting Assay

In order to investigate antimicrobial activity of halocidin, the presentinventors performed colony counting assay. Particularly, adjusted thefinal concentration of the peptide to 5 μg/ml by mixing the peptide andMRSA strain or MDRPA strain of mid-log phase in sterilized 10 mM sodiumphosphate buffer (pH 7.4) containing 0.3 mg/id of TSB powder. Adjustedthe final volume of the above mixture to 100 μl, and then let it bepre-reacted in a 37° C. shaking water bath for 5 and 15 minutes each.Collected 20 μl of pre-reacted solution and loaded thereof onto 1.5%bacto-agar plate (Difco). Induced reaction for overnight and counted thenumber of formed colonies on the above plate, from which measured theantimicrobial activity. Used magainin 1 and buforin 2 for comparativegroup, and 0.01% acetic acid for control group.

As a result, comparative group and control group hardly showedantimicrobial activity to MRSA strain and MDRPA strain while 18Hc,di-18Hc and halocidin showed high antimicrobial activity from 5 minutesafter reaction began, which was continued until the 15 minutes afterreaction. Especially, di-18Hc showed the highest antimicrobial activityand 18Hc and halocidin followed in order (FIG. 10). From the aboveresults, the present inventors confirmed that 18Hc monomer or dimer,constituents of halocidin, has higher antimicrobial activity thanhalocidin itself.

<3-3> Hemolytic Assay

In order to investigate antimicrobial activity of halocidin, the presentinventors performed hemolytic assay. Particularly, mixed 20 μl ofpeptide diluted to 100, 50, 25, 12.5, 6.25, 3.125 μg/ml and 180 μl of2.5% (V/V) human erythrocytes in PBS. Used melittin (Sigma) and clavaninAK, a congener in which clavanin A residue was substituted, forcomparative group, and 0.01% acetic acid for control group. Afterreacting the mixture at 37° C. for 30 minutes, added 600 μl of PBS intoeach tube. Centrifuged the solution at 10,000 g for 3 minutes andseparated supernatants. Measured OD at 540 nm and calculated thehemolytic activity (%) according to the below <Mathematical Formula 1>.

$\begin{matrix}{{{Hemolytic}\mspace{14mu}{Activity}\mspace{11mu}(\%)} = {\frac{\begin{matrix}{{Sample}\mspace{14mu} A_{540}\bullet} \\{{Control}\mspace{14mu}{Group}\mspace{14mu} A_{540}}\end{matrix}}{\begin{matrix}{{100\%\mspace{14mu}{Comparative}\mspace{14mu}{Group}\mspace{14mu} A_{540}} -} \\{{Control}\mspace{14mu}{Group}\mspace{14mu} A_{540}}\end{matrix}} \times 100}} & {< {{Mathematical}\mspace{14mu}{Formula}\mspace{14mu} 1} >}\end{matrix}$

As a result, the hemolytic activity of each peptide di-18Hc, 18Hc, 15Hc,di-15Hc and halocidin was proved to be 18%, 9%, 0% and 0% (FIG. 11).Therefore the hemolytic activity of di-18Hc was the highest.

<3-4> Comparing Antimicrobial Activity to Gram Negative Bacteria

The present inventors confirmed the antimicrobial activity of halocidinto Gram-negative bacteria with radical diffusion assay. Particularly,investigated antimicrobial activities of 15Hc, 18Hc and halocidin toGram negative bacteria such as Pseudomonas aeruginosa, Salmonellacholerasuis, Salmonella parotyphi A, E. coli K112 and E. coli DH5α. Atthat time, used buforin 2 and magainin 1 for comparative group.

As a result, 15Hc and 18Hc hardly showed or had minimum antimicrobialactivity even though the concentration of peptide increased. However,halocidin showed almost the same level of antimicrobial activity asmagainin 1 and buforin 2, which were the comparative group, or higherantimicrobial activity than comparative group according to the kinds ofbacteria (FIG. 12).

The present inventors performed radical diffusion assay again for(18-1)Hc, di-(18-1)Hc, 18Hc and di-18Hc with the same method as theabove. As a result, di-(18-1)Hc and di-18Hc, which were in dimer form,showed higher antimicrobial activity than (18-1)Hc and 18Hc, which werein monomer forms, though it varied upon the kinds of bacteria (FIG. 13).

The present inventors performed radical diffusion assay for (18-2)Hc,di-(18-2)Hc, (18)Hck and di-(18)Hck with the same method as the above.As a result, di-(18-2)Hc and di-(18)Hck, which were in dimer forms,showed higher antimicrobial activity than (18-2)Hc and (18)Hck, whichwere in monomer forms (FIG. 14).

Based on the above results, the present inventors confirmed thathalocidin had high antimicrobial activity to Gram-negative bacteria andshowed the highest activity when monomers, subunits of halocidin, werein dimer forms.

<3-5> Comparing Antimicrobial Activity to Gram Positive Bacteria

The present inventors investigated the antimicrobial activity ofhalocidin to Gram-positive bacteria. Particularly, performed radicaldiffusion assay with the same method as the above Example 1 to confirmthe antimicrobial activity of each 15Hc, 18Hc, halocidin, buforin 2 andmagainin 1 to Gram-positive bacteria such as Staphylococcus aureus,Micrococcus luteus, Enterococcus faecalis, Bacillus subtilus and MRSA.

As a result, just halocidin showed high antimicrobial activity. 15Hc and18Hc were proved to have low antimicrobial activity (FIG. 15).

The present inventors performed radical diffusion assay again for(18-1)Hc, di-(18-1)Hc, 18Hc and di-18Hc with the same method as theabove. As a result, di-(18-1)Hc and di-18Hc, which were in dimer forms,showed higher antimicrobial activity than (18-1)Hc and 18Hc, which werein monomer forms, though it varied upon the kinds of bacteria (FIG. 16).

The present inventors performed radical diffusion assay for (18-2)Hc,di-(18-2)Hc, (18)Hck and di-(18)Hck with the same method as the above.As a result, di-(18-2)Hc and di-(18)Hck, which were in dimer forms,showed higher antimicrobial activity than (18-2)Hc and (18)Hck, whichwere in monomer forms (FIG. 17).

Based on the above results, the present inventors confirmed thathalocidin had high antimicrobial activity to Gram-positive bacteria andshowed the highest activity when monomers, subunits of halocidin, werein dimer forms.

<3-6> Antimicrobial Activity According to pH

In order to confirm if halocidin having high antimicrobial activity tobacteria still keeps the activity under the low pH condition in vivo,the present inventors investigated the change of antimicrobial activityaccording to pH by radical diffusion assay. Particularly, adjusted thepH of media to 7.4, 6.5 and 5.5 respectively by adding HCl, which waschecked with a pH meter, and performed radical diffusion assay with thesame method as the above Example 1 to investigate the antimicrobialactivity to MRSA and Enterococcus faecalis.

As a result, it was confirmed that di-18Hc, di-(18-1)Hc and di-k(18+1)Hckept the same antimicrobial activity even under low pH condition, pH5.5, which was similar condition to the environment of epithelialtissue, urethra, intravagina, etc, as that under pH 7.4(Table 4).

TABLE 4 Enterococcus Peptide MRSA faecalis pH = 7.4 Halocidin >64 >64K(18 + 1)Hc 16-32  8-16 di-(18 − 1)Hc  8-16  8-16 di-18Hc  8-16 2-4di-K(18 + 1)Hc 2-4 2-4 P18  8-16 32-64 Magainin 1 >64 >64 Buforin2 >64 >64 Secrofin A >64 >64 pH = 6.5 Halocidin >64 >64 K(18 + 1)Hc16-32 16-32 di-(18 − 1)Hc 2-4 4-8 di-18Hc 2-4 4-8 di-K(18 + 1)Hc 2-4 2-4P18 32-64 >64 Magainin 1 >64 >64 Buforin 2 >64 >64 Secrofin A >64 >64 pH= 5.5 Halocidin >64 >64 K(18 + 1)Hc 32-64 16-32 di-(18 − 1)Hc  8-16 2-4di-18Hc 4-8 2-4 di-K(18 + 1)Hc 2-4 2-4 P18 >64 >64 Magainin 1 >64 >64Buforin 2 >64 >64 Secrofin A >64 >64<3-7> Antimicrobial Activity According to Base

The present inventors confirmed if halocidin still had antimicrobialactivity under the strong basic condition in vivo. Particularly, changedthe basic condition of media by adding 100 mM NaCl, 150 mM NaCl and 200mM NaCl respectively, and investigated the antimicrobial activity toMRSA and Enterococcus faecalis by radical diffusion assay.

As a result, it was confirmed that di-18Hc, di-(18-1)Hc and di-k(18+1)Hckept the same antimicrobial activity even under strong basic condition(200 mM) as that under non-basic condition (Table 5).

TABLE 5 Enterococcus Peptide MRSA faecalis NaCl Halocidin >64 >64 100 mMK(18 + 1)Hc 16-32 16-32 di-(18 − 1)Hc 4-8  8-16 di-18Hc  8-16 2-4di-K(18 + 1)Hc 4-8 2-4 P18 >64 >64 Magainin 1 >64 >64 Buforin 2 >64 >64Secrofin A >64 >64 NaCl Halocidin >64 >64 150 mM K(18 + 1)Hc 16-32 16-32di-(18 − 1)Hc  8-16  8-16 di-18Hc  8-16 2-4 di-K(18 + 1)Hc 4-8 2-4P18 >64 >64 Magainin 1 >64 >64 Buforin 2 >64 >64 Secrofin A >64 >64 NaClHalocidin >64 >64 200 mM K(18 + 1)Hc 32-64 16-32 di-(18 − 1)Hc  8-16 8-16 di-18Hc 16-32 2-4 di-K(18 + 1)Hc 4-8 2-4 P18 >64 >64 Magainin1 >64 >64 Buforin 2 >64 >64 Secrofin A >64 >64

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. A peptide separated from tunicate and comprising amino acid sequencerepresented by chemical formula I:W₁X₂B′₃U₄X₅X₆B₇B₈U₉X₁₀B′₁₁C₁₂U₁₃B₁₄U₁₅X₁₆X₁₇U₁₈ (SEQ ID NO: 11)  (I)wherein, W represents tryptophan; X, each variable of which X₂, X₅, X₆,X₁₀, X₁₆ and X₁₇ is individually selected from an amino acid residueselected from the group consisting of tyrosine, valine, isoleucine,leucine, methionine, phenylalanine and tryptophan; B represents an aminoacid residue selected from the group consisting of arginine, lysine andhistidine; B′ represents an amino acid residue selected from the groupconsisting of arginine, lysine and histidine or from a group consistingof asparagine and glutamine; C is Cysteine; U represents an amino acidresidue selected from the group consisting of glycine, serine, alanineand threonine.
 2. The peptide as set forth in claim 1, wherein thetunicate is Halocynthia aurantium.
 3. The peptide as set forth in claim1, wherein the peptide comprises amino acid sequence SEQ. ID No:
 1. 4. Apeptide comprising an amino acid sequence represented by chemicalformula II:U₄X₅X₆B₇B₈U₉X₁₀B′₁₁C₁₂U₁₃B₁₄U₁₅X₁₆X₁₇U₁₈ (SEQ ID NO: 13)  (II) wherein,U represents an amino acid residue selected from a group consisting ofglycine, serine, alanine and threonine; X, each variable of which X₅,X₆, X₁₀, X₁₆ and X₁₇ is individually selected from an amino acid residueselected from the group consisting of tyrosine, valine, isoleucine,leucine, methionine, phenylalanine and tryptophan; B represents an aminoacid residue selected from the group consisting of arginine, lysine andhistidine; and B′ represents an amino acid residue selected from thegroup consisting of arginine, lysine and histidine or from a groupconsisting of asparagine and glutamine.
 5. The peptide as set forth inclaim 4, wherein the peptide comprises an amino acid sequencerepresented by SEQ ID NO: 15 in which U₄ is alanine, X₅ is leucine, X₆is leucine, B₇ is histidine, B₈ is histidine, U₉ is glycine, X₁₀ isleucine, B′₁₁ is asparagine, C₁₂ is cysteine, U₁₃ is alanine, B₁₄ islysine, U₁₅ is glycine, X₁₆ is valine, X₁₇ is leucine and U₁₈ isalanine.
 6. A peptide dimer comprising an amino acid sequencerepresented by chemical formula III: wherein each peptide of the dimeris represented by chemical formula I (SEQ ID NO: 11) and the peptidesare joined at the cysteine site by disulfide bond;


7. A peptide dimer comprising an amino acid sequence represented byformula IV: wherein each peptide of the dimer is represented by chemicalformula II (SEQ ID NO: 13), and the peptides are joined at the cysteinesite by disulfide bond;


8. A peptide dimer comprising an amino acid sequence represented byformula V: wherein one peptide of the dimer is represented by chemicalformula I (SEQ ID NO: 11) and another peptide of the dimer isrepresented by chemical formula II (SEQ ID NO: 13), and the peptides arejoined at the cysteine site by disulfide bond;


9. An antimicrobial agent comprising a peptide comprising the chemicalformula I of claim 1 as an active ingredient.
 10. An antimicrobial agentcomprising a peptide comprising the chemical formula II of claim 4 as anactive ingredient.
 11. An antimicrobial agent comprising a peptide dimercomprising the chemical formula III of claim 6 as an active ingredient.12. An antimicrobial agent comprising a peptide dimer comprising thechemical formula IV of claim 7 as an active ingredient.
 13. Anantimicrobial agent comprising a peptide dimer comprising the chemicalformula V of claim 8 as an active ingredient.